US 7910723 B2
Methods and compositions inhibit the growth of cancer cells by selectively down-regulating the expression of an IG20 splice variant including MADD. Specific knock-down of MADD splice variant resulted in the apoptosis of cancer cells. Interfering RNAs including small hairpin RNAs (shRNA) to down-regulate MADD expression in vivo are disclosed. Inhibition of MADD phosphorylation by Akt results in activation of cancer cell death. Down-regulation of MADD expression results in switching to apoptotic mode due to lack of MAPK activation upon TNF-α-based induction.
1. An isolated siRNA nucleic acid that selectively down-regulates the expression of at least one splice variant of an IG20 (Insulinoma-Glucagonoma) gene, wherein the splice variant is MADD represented by the cDNA sequence of SEQ ID NO: 1, and wherein not all isoforms of IG20 are knocked down.
2. The nucleic acid of
Xsense —hairpin loop—Xanti-sense
wherein X comprises a nucleic acid sequence CGGCGAATCTATGACAATC (SEQ ID NO: 4).
3. The nucleic acid of
4. The nucleic acid of
5. The nucleic acid of
6. A pharmaceutical composition consisting essentially of the nucleic acid of
7. The pharmaceutical composition of
8. The nucleic acid of
9. The nucleic acid of
This application is a continuation-in-part application under 35 U.S.C §365 of International Application No. PCT/US2007/060712, filed Jan. 18, 2007, which claims priority to U.S. Ser. No. 60/760,321, filed Jan. 19, 2006, the contents of which are hereby incorporated by reference in their entirety.
The United States government has certain rights in this invention pursuant to a grant number 5R01CA107506 from the National Institutes of Health.
In eukaryotes, many genes undergo alternative splicing and encode multiple isoforms leading to the expression of related proteins that have distinct biochemical as well as biological features. The IG20 (Insulinoma-Glucagonoma) is one such gene that undergoes alternative splicing and encodes at least four different splice variants (SVs), namely IG20pa, MADD/DENN, IG20-SV2 and DENN-SV. These four IG20-SVs are distinguished by differential splicing of exons 13L and 16. Upon comparison to IG20pa, the splice variants MADD, IG20-SV2 and DENN-SV lack the expression of exon 16 or 13L, or both respectively. All four IG20-SVs express an N-terminal leucine zipper and a C-terminal death-domain homology region.
The IG20 gene plays an important role in cancer cell proliferation, apoptosis and survival, most likely through its effects on MAP kinase activation and other cell signaling pathways. Additionally, it plays an important role in neurotransmission, neurodegeneration and guanine nucleotide exchange. How IG20 is involved in these divergent functions is not yet fully known.
Expression of the IG20 gene is relatively high in cancer cells and tissues as compared to the levels of expression in their normal counterparts. While MADD and DENN-SV are constitutively expressed (DENN-SV is over-expressed relative to other SVs in cancer), expression of IG20pa and IG20-SV2 appears to be regulated in that they may or may not be expressed in certain cells. Gain of function studies through expression of individual IG20-SVs in HeLa cells showed that MADD and IG20-SV2 variants have little or no effect on cell proliferation and induced apoptosis. IG20pa increased susceptibility to both extrinsic and intrinsic apoptotic stimuli, and suppressed cell proliferation and DENN-SV conferred resistance to induced apoptosis and enhanced cell proliferation. Thus IG20pa and DENN-SV acted like a “tumor suppressor” and an “oncogene” respectively.
Knock-down of all endogenous IG20-SVs, using anti-sense oligonucleotides, resulted in spontaneous apoptosis of cancer cells in vitro and in vivo, but not in normal cells. Since different splice variants have different functions and the function of IG20 gene may vary depending upon the cell type, it is prudent to develop modalities that allow knockdown of specific isoforms to achieve the desired effects including altering cell growth, apoptosis, neuron-transmission. Such isoform-specifc knock-down has not yet been demonstrated. In addition, the contrasting effects of IG20-SVs noted from gain of function studies can be clarified by knocking-down individual endogenous IG20-SVs and determining the consequent effects. This poses several challenges because various IG20-SVs differ from each other only by the differential expression of very short exons 13L (130 base pairs) and 16 (60 base pairs). Therefore, knock-down of specific splice variants of IG20 gene is difficult because of availability of very short target sequences that are differentially expressed in different splice variants and is achieved through the use of specially designed small hairpin RNA molecules (shRNA) disclosed herein.
Among the IG20 isoforms, MADD-SV acts as a negative regulator of caspase-8 activation and is necessary and sufficient for cancer cell survival. Abrogation of MADD-SV, but not the other IG20-SVs rendered cancer cells more susceptible to spontaneous as well as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. Also, the expression of MADD-SV alone in the absence of endogenous IG20-SVs is sufficient to prevent spontaneous apoptosis. MADD-SV plays a predominant role in cancer cell survival by acting as a negative regulator of caspase-8 activation. This profound effect is not due to direct association of MADD-SV with caspase-8. One possibility is that binding of MADD-SV to the receptor activates prosurvival pathways like MAP kinase or NF-kB pathway thereby antagonizing caspase-8 activation and leading to cancer cell survival.
The mitogen-activated protein kinases (MAPKs) are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate several important and critical cellular functions required for cell homeostasis like metabolism, cell cycle progression, expression of cytokines, motility and adherence. Hence MAP kinases influence cell survival, proliferation, differentiation, development and apoptosis. Extracellular stimuli such as cytokines, growth factors and environmental stresses lead to the sequential activation of a signaling cascade composed of MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and MAPK. The three main members of MAPK family are extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun-amino-terminal kinase (JNK) and p38.
The ERK pathway is a drug target for cancer chemotherapy of all the mammalian MAPK pathways since in approximately, one-third of all human cancers there is deregulation of the pathway leading to ERK activation. When activated, ERK1/2 phosphorylates several nuclear and cytoplasmic substrates involved in a multitude of cellular processes, including transcriptional factors, signaling proteins, kinases and phosphatases, cytoskeletal proteins, apoptotic proteins and proteinases. Even though the ERK pathway can be activated by numerous extracellular signals, the pathways whereby cytokines and growth factors activate ERK signaling are of particular relevance to cancer. TNF-α, a cytokine rich in tumor stroma binds to TNFR1 (TNF receptor1) present on cancer cells and potently activates ERK MAPK. In the absence of MADD this pro-survival signaling pathway can be converted into an apoptotic signaling pathway leading to cancer cell death even in the absence of protein synthesis inhibitor like cycloheximide.
The mitogen-activated kinase activating death domain protein (MADD) is expressed at very low levels in a variety of tissues and organs under physiological conditions. However, it is over-expressed in many types of human tumors and tumor cell lines. Enforced expression of exogenous MADD has no apparent effect on cell survival, but knockdown of endogenous MADD can lead to spontaneous as well as enhanced tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) induced apoptosis, indicating that MADD is necessary for cancer cell survival. Furthermore, MADD contributes to the resistance of PA-1 ovarian carcinoma cells to TRAIL-induced apoptosis.
The extrinsic apoptotic pathway is initiated by upon death ligand (e.g. TRAIL), binding to its cognate death receptors, which undergo trimerization and recruit FADD and subsequent caspase-3 activation. MADD can bind to DRs thereby inhibit DISC formation. Knock-down of MADD causes spontaneous as well enhanced TRAIL-induced apoptosis. Thus, MADD can contribute to cell survival by blocking activation of the extrinsic apoptotic pathway.
The intrinsic pathway is initiated when a death signal induces the release of mitochondrial pro-apoptotic proteins such as cytochrome c, mitochondrial apoptosis-inducing factor and Smac/Diablo. Cytochrome c forms a complex with Apaf-1 and procaspase-9 resulting in the activation of caspase-9, while Smac/Diablo can associate with inhibitor of apoptosis proteins (IAPs) and counteract their inhibitory effects. The intrinsic pathway is regulated by the Bcl-2 family members. For example, in response to proapoptotic stimuli, the cytosolic Bax and Bad translocate to mitochondria leading to cytochrome c release into the cytosol. In contrast, Bcl-2 and Bcl-xL can associate with Bax and Bad to prevent cell death.
Apoptotic factors are controlled by a complex signaling system. For example, the pro-apoptotic function of Bad is regulated by phosphorylation that prevents Bad from inducing apoptosis. p53 function in regulating apoptosis is related to its phosphorylation status. The apoptosis repressor protein function is enabled by its phosphorylation at threonine-149. Given the important role of MADD in controlling apoptosis, there is a question whether its function is constitutive or is also regulated by other signals.
Protein kinase B (i.e. Akt) promotes cell survival by phosphorylating a variety of apoptosis-related factors containing the consensus sequence RXRXX(S/T). Akt phosphorylates mouse double minute 2 and enhances its ability to degrade p53. Akt can phosphorylate a number of apoptosis related proteins such as caspase-9, Bad, IKKα, Forkhead transcription factor, mdm2 and Yap, and play a critical role in TSC1/2, Rheb/mTOR signalling pathway. Interestingly, PI3K can also activate RAS function leading to the activation of the MAPK prosurvival pathway, in which MADD plays an important role.
Role of IG20 splice variants including MADD in cancer therapeutics was analyzed. Disruption of MADD phosphorylation by Akt results in death of cancer cells. siRNA molecules that specifically target IG20 splice variants are useful as pharmaceutical therapeutics against cancer.
Methods and compositions selectively down-regulate the expression of a particular IG20 gene splice variant and thereby promote cancer cell death in cells that express the particular splice variant. For example, the MADD splice variant of IG20 gene was down regulated by the use of interfering RNA sequences that specifically down regulate MADD splice variant and the down regulation was sufficient to cause apoptotic death of cancer cells. Endogenous MADD-SV, a pro-survival IG20 splice variant plays an essential role in activating prosurvival ERK MAPK pathway upon TNF-stimulation thereby antagonizing caspase-8 activation and preventing cell apoptosis.
Using shRNAs that specifically target exon 15 that is expressed in all isoforms of IG20 and designated Mid, or exons 13L and 16 that are differentially expressed in IG20-SVs, the various splice variants of IG20 gene were selectively knocked-down in HeLa and PA-1 cells. Knock down of MADD (one of the IG20 splice variants) resulted in spontaneous apoptosis and this effect was reversible by re-expressing the MADD protein. This result indicated that MADD is required and sufficient for the survival of cancer cells. Further, knock down of MADD rendered cells more susceptible to TRAIL induced apoptosis. The increased apoptosis was associated with increased caspase-8 and caspase-3 activation. The results presented herein demonstrate that knock-down of MADD causes a significant increase in spontaneous as well as TRAIL induced cell death of cancer cells, and support the conclusion that it is MADD, and not the other three isoforms (i.e. DENN-SV, IG20-SV2 and IG20) that is required for cancer cell survival.
MADD is phosphorylated by Akt and phosphorylated MADD binds to the death receptor (DR) and prevent activation of the extrinsic apoptotic pathway. While non-phosphorylated MADD loses its binding to DR, it gains binding to 14-3-3 causing Bax disassociation from 14-3-3 and translocation to mitochondria. While loss of MADD binding to DR leads to spontaneous activation of the extrinsic pathway, Bax translocation leads to the activation of the intrinsic pathway. Moreover, TRAIL treatment causes accumulation of non-phosphorylated Akt as well as MADD, which facilitates caspase-8 activation on one hand, and Bax translocation to mitochondria on the other. These results show that MADD, depending upon Akt phosphorylation, regulates both extrinsic and intrinsic apoptotic pathways.
MADD-SV of the IG20 gene acts as a negative regulator of caspase-8 activation. The molecular mechanism of action of MADD-SV in regulating caspase-8 activation is presented herein. Down-modulation of MADD-SV facilitated caspase-8 activation without affecting TNF-α induced NF-kB activation. The ability of TNF-α, TRAIL, LPS and EGF to activate MAP kinases in the presence or absence of various IG20-SVs was analyzed. Knock-down of all IG20-SVs or MADD-SV alone resulted in a dramatic loss in ERK activation only upon TNF-α treatment. Similar effects were not seen when cells were stimulated with other ligands. Also, knock-down of MADD-SV had little or no effect on the activation of other MAP kinases such as JNK and p38 upon TNF-α treatment. Over-expression of ShRNA resistant exogenous MADD-SV followed by knock-down of all endogenous IG20-SVs restored MAP kinase activation and unequivocally demonstrated that MADD-SV is necessary and sufficient for TNF-α induced ERK activation. Down-modulation of MADD-SV, which abrogates ERK activation, rendered cancer cells more susceptible to TNF-α induced apoptosis as evidenced by enhanced caspase-3 activation and decreased activation of transcriptional factor p90RSK. These results indicate that down-modulation of endogenous MADD-SV positively regulates caspase-8 activation by abrogating ERK MAP kinase pathway.
A method of selectively inhibiting a splice variant of an IG20 (Insulinoma-Glucagonoma) gene, includes the steps of:
A suitable splice variant is MADD and a suitable agent is a molecule selected from the group that includes siRNA, shRNA and an anti-sense molecule against the IG20 splice variant.
A method of specifically down-regulating the expression of a splice variant of an IG20 (Insulinoma-Glucagonoma) gene includes the steps of:
A nucleic acid molecule includes a nucleotide sequence of CGGCGAATCTATGACAATC (SEQ ID NO: 4) or a transcribed product thereof that is sufficient to knock-down the expression of MADD splice variant or an allelic variant or a mutant thereof.
A method of inhibiting the growth of a cancer cell includes the steps of:
A suitable nucleic acid molecule targets exon 13L of the MADD splice variant.
A suitable nucleic acid molecule is selected from a group that includes siRNA, shRNA and anti-sense molecule against the MADD splice variant of IG20 that targets a nucleotide sequence of MADD selected from a group that includes CGGCGAATCTATGACAATC (SEQ ID NO: 4), allelic variation thereof, polymorphisms thereof, and a genetic mutation thereof.
A method of regulating the growth of a cancer cell includes the steps of:
A method of increasing apoptotic cell death in a cancer cell includes the steps of:
In an aspect, the apoptotic cell death is effected by a caspase. The apoptotic cell death is also induced by a TNF-related apoptosis inducing ligand (TRAIL).
A suitable nucleic acid molecule capable of inducing apoptotic cell death includes a nucleotide sequence CGGCGAATCTATGACAATC (SEQ ID NO: 4) or an RNA equivalent thereof.
A pharmaceutical composition consists essentially of a nucleic acid sequence capable of selectively inhibiting the expression of a MADD splice variant in a cancer cell. The pharmaceutical composition includes a nucleic acid whose nucleotide sequence includes CGGCGAATCTATGACAATC (SEQ ID NO: 4) or an RNA equivalent thereof.
A vector includes a nucleic acid sequence capable of selectively inhibiting the expression of a MADD splice variant in a cancer cell, wherein the nucleic acid sequence includes CGGCGAATCTATGACAATC (SEQ ID NO: 4).
A cell includes an exogenous molecule of an interfering RNA, wherein the RNA molecule specifically down-regulates the expression of a MADD splice variant of an IG20 gene. The cell may be a cancer cell or a cell predisposed or likely to become cancerous.
An isolated nucleic acid molecule encodes a short interfering RNA, the nucleic acid includes the structure:
wherein X includes a nucleic acid sequence CGGCGAATCTATGACAATC (SEQ ID NO: 4). A suitable shRNA sequence is generated by a nucleic acid sequence CGGCGAATCTATGACAATCTTCAAGAGAGATTGTCATAGATTCGCCG (SEQ ID NO: 5), wherein the hairpin loop region is from positions 20-28 (shown as underlined). The nucleic acids may be synthetic.
An isolated RNA molecule includes a nucleic acid sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO: 6).
A method of inducing cell death in a cancer cell includes:
Suitable agents include small molecules, peptides and peptide derivatives. For example, an agent is a MADD peptide that includes one or more phosphorylation sites for Akt. In some aspects, the agent is administered as an adjuvant therapy along with chemotherapy or radiation therapy. In some aspects, the cancer cell is resistant to TRAIL-induced apoptosis.
A method of identifying an inhibitor of the phosphorylation of MADD by Akt includes:
An antibody is generated against a peptide epitope selected from CRQRRMpSLRDDTS (SEQ ID NO: 7) (S-70), GSRSRNSpTLTSL (SEQ ID NO: 8) (T-173), and KRKRSPpTESVNTP (SEQ ID NO: 9) (T-1041), wherein Mp or Sp or Pp is a phosphorylated amino acid. In some aspects, the testing is performed in the presence of phosphorylated MADD.
A purified mutant MADD polypeptide includes one or more mutations at amino acid positions 70, 173, and 1041.
In an aspect the MADD polypeptide includes an amino acid sequence of SEQ ID NO: 3, wherein the amino acids at positions 70, 173, and 1041 are all mutated such that Akt does not phosphorylate MADD.
Methods and compositions described herein relate selective down-regulation of a specific IG20 splice variant to promote apoptosis of cancer cells. Through the use of specific small hairpin RNA (shRNA) molecules, knock-down of an IG20 splice variant, e.g., MADD is demonstrated. This knock-down of MADD splice variant in cancer cells resulted in apoptotic cell death. Cell death of cancer cells is further characterized by activation of caspases that are responsible for apoptotic cell death.
Down-regulation of MADD splice variant is accomplished by a number of ways. For example knock-down of MADD splice variant is accomplished through shRNA, siRNA, anti-sense expression, small-molecules that specifically lower the RNA levels of MADD or inactivate the activity of MADD protein, and synthetic peptide nucleic acid (PNA) oligomers (e.g., containing repeating N-(2-aminoethyl)-glycine units linked by peptide bonds).
Agents capable of down-regulating MADD expression are delivered directly to tumors, administered by a viral vector capable of transcribing and producing an interfering RNA (RNAi) molecule, liposome, and as pharmaceutical compositions. shRNAs and siRNAs can also be delivered as synthetic molecules.
The IG20 gene is over-expressed in human tumors and cancer cell lines, and encodes at least four splice variants (SVs) namely, IG20 (here referred to as IG20pa), MADD, IG20-SV2 and DENN-SV. Gain of function studies showed that IG20-SVs can exhibit diverse functions and play a critical role in cell proliferation and apoptosis. Expression of exogenous IG20pa or DENN-SV rendered cells either susceptible or resistant to induced apoptosis, respectively; while MADD and IG20-SV2 had no apparent effect.
The contrasting effects of the IG20-SVs in a more physiologically relevant system are analyzed herein by using exon-specific shRNAs to selectively knock-down specific IG20-SVs. Knock-down of all IG20-SVs resulted in spontaneous apoptosis of HeLa and PA-1 cells. Simultaneous knock-down of all the splice variants of IG20 may not be therapeutically as effective as selective knock-down because down-regulation of all the splice variants result in unexpected and undesirable outcomes because different splice variants exhibit different physiologically relevant functions such as cell growth, cell growth inhibition, neurotransmission and the like. Moreover, the IG20 gene through its splice variants, can exert different functional effects on different tissues (e.g. neurotransmission in neuronal cells). Also, knock down of all splice variants may be harmful as evidenced by the inability of IG20 gene knockout mice to survive. Therefore, knock-down of select isoforms facilitates induction of the intended effect and minimizes harmful effects, e.g., death of normal cells.
Knock-down of MADD can render cells susceptible to spontaneous apoptosis but had no discernible effect on cell proliferation, colony size, or cell cycle progression. The utility of shRNAs for selective knock-down of particular IG20-SVs is demonstrated. MADD isoform expression is required for cancer cell survival, and therefore the methods and compositions disclosed herein are therapeutically valuable in targeting specific IG20-SVs to reduce cancer growth and thereby promoting selective cancer cell death.
MADD abrogation, in addition to causing spontaneous apoptosis, also enhances TRAIL-induced apoptosis. MADD interacts with the death receptors (DRs) but not with either the FADD or caspase-8, and the spontaneous as well as enhanced TRAIL induced apoptosis result from activation of caspase-8 at the DRs without an apparent increase in the recruitment of DISC components. Under physiological conditions, MADD acts as a negative regulator of caspase-8 activation.
Gain of function studies using exogenous IG20-SVs showed that MADD and IG20-SV2 have little or no effect on cell proliferation and susceptibility to induced apoptosis. However, IG20pa rendered cells highly susceptible to apoptosis induced by different death signals including TRAIL, and suppressed cell proliferation. In contrast, it is found that DENN-SV was over expressed in tumor tissues and cancer cell lines, and expression of exogenous DENN-SV confers resistance to apoptosis and enhance cell proliferation.
Down-modulation of select combinations of IG20-SVs using siRNAs is demonstrated herein. Synthetic siRNA duplexes or expressed shRNAs binds to the target mRNA and lead to its degradation. Specific and the most effective shRNAs against IG20-SVs were identified by screening 5 different shRNAs targeting all isoforms, and 2 each targeting exons 13L and 16 and cloned into a lentivirus vector. Use of lentivirus resulted in stable expression of shRNAs that is detected through GFP expression. Expressed shRNA down-modulated the targeted IG20-SVs as early as 24 hours post transduction and lasted at least for 15 days.
Significant increase in spontaneous apoptosis of HeLa cells with knock-down of all IG20-SVs was noted when assayed for nuclear condensation and mitochondrial depolarization; hallmarks of apoptosis. Earlier studies failed to identify the specific IG20-SV responsible, for cancer cell survival.
Significant spontaneous apoptosis was observed at 72 hours although the relevant IG20-SV transcripts were down-modulated at 24 hours. This is likely due to persistence of pre-formed proteins, although the possibility that this duration is required for either accumulation of apoptotic, or down-modulation of anti-apoptotic, molecules cannot be ruled out.
Isolated siRNA nucleic acids that selectively down-regulate the expression of a splice variant of an IG20 (Insulinoma-Glucagonoma) gene, wherein the splice variant is MADD are disclosed. In an embodiment, the nucleic acid encodes a short interfering RNA, that includes the structure:
wherein X includes or consists essentially of a nucleic acid sequence CGGCGAATCTATGACAATC (SEQ ID NO: 4).
In an embodiment, the siRNA nucleic acid sequence is CGGCGAATCTATGACAATCTTCAAGAGAGATTGTCATAGATTCGCCG (SEQ ID NO: 5), wherein the hairpin loop region is from positions 20-28 of the sequence. The hairpin loop region may contain any suitable sequence. In an embodiment, siRNA or shRNA molecules disclosed herein are synthetic and may include nucleic acid modifications to enhance stability.
RNA molecules that include a nucleic acid sequence CGGCGAAUCUAUGACAAUC (SEQ ID NO: 6) are disclosed. RNA molecules that are transcribed in vitro or in vivo, e.g., in a cancer cell or tumors are also included.
A method of specifically down-regulating the expression of a splice variant of an IG20 (Insulinoma-Glucagonoma) gene includes:
Specifically down-regulating may refer to a substantial down-regulation for example, more than 90% or 95% reduction of the endogenous MADD transcript. In some aspects, the acid molecule molecules specifically target exon 13L of the MADD splice variant. Thus, based on the target sequence and the siRNA sequences disclosed herein a variety of specific siRNA or shRNA nucleic acid molecules are designed for therapeutics.
Nucleic acid molecules that consist essentially of a nucleotide sequence CGGCGAATCTATGACAATC (SEQ ID NO: 4) or a transcribed product thereof are sufficient to knock-down the expression of MADD splice variant or an allelic variant or a mutant thereof. Natural variations of MADD including specific SNPs, allelic variants that may appear in one or more of sub-groups of the cancer types are also targeted by the nucleic acid molecules disclosed herein. Accordingly, one or more nucleic acid changes are adopted by those skilled in the art.
A method of inhibiting the growth of a cancer cell includes:
In some aspects, the nucleic acid molecule is selected from a group that includes siRNA, shnRNA and anti-sense molecule against the MADD splice variant of IG20 that targets a nucleotide sequence of MADD selected from CGGCGAATCTATGACAATC (SEQ ID NO: 4), allelic variation thereof, polymorphisms thereof, and a genetic mutation thereof.
A method of regulating the growth of a cancer cell includes:
A method of increasing apoptotic cell death in a tumor includes:
In some aspects, the apoptotic cell death is affected by a caspase. In an aspect, the apoptotic cell death is induced by a TNFα-related apoptosis inducing ligand (TRAIL). For example, in an aspect, the down-regulation of MADD abrogates prosurvival function of TNFα-induced MAPK activation, such that apoptosis is triggered when MADD is not available for MAPK phosphorylation. The tumor may be a solid tumor. TNFα usually activates MAPK in tumor stroma and facilitates neovasculogenesis required for tumor survival (nutrition). However, upon MADD knock-down this prosurvival function is switched to apoptosis.
A method of selectively inhibiting a splice variant of an IG20 (Insulinoma-Glucagonoma) gene includes:
Pharmaceutical compositions consisting essentially of the nucleic acid, e.g., CGGCGAATCTATGACAATC (SEQ ID NO: 4) or an RNA equivalent thereof capable of selectively inhibiting the expression of a MADD splice variant in a cancer cell are disclosed. Pharmaceutical compositions may also include pharmaceutical carriers, incipients and any other ingredients suitable for nucleic acid or peptide delivery.
Vectors that include the nucleic acid molecule having a sequence CGGCGAATCTATGACAATC (SEQ ID NO: 4) capable of selectively inhibiting the expression of a MADD splice variant in a cancer cell are disclosed. The vectors are capable of delivering the nucleic acid molecules to a plurality of tumor cells. The vectors may also be part of a liposome or any suitable carrier.
Cells including host cells, transformed cells, transformed tumor cells include the nucleic acid molecule e.g., having a sequence CGGCGAATCTATGACAATC (SEQ ID NO: 4) or an RNA equivalent thereof, wherein the nucleic acid specifically down-regulates the expression of a MADD splice variant of an IG20 gene.
A method of inducing cell death in a cancer cell includes:
In an embodiment, an agent is selected from small molecules, peptides and peptide derivatives. For example, a suitable agent is a MADD peptide that includes one or more phosphorylation sites for Akt, and the MADD-derived peptide is capable of interacting with Akt and serves as a phosphorylation substrate, thereby competing with the endogenous MADD and prevents MADD phosphorylation by Akt. Suitable MADD-derived peptides or polypeptides include for example one or more of the phosphorylation sites of MADD and in some aspects include all 3 phosphorylation sites. In an aspect, the MADD-derived peptides compete for Akt phosphorylation and thereby prevents or minimizes MADD phosphorylation by Akt. In some aspects, the MADD-derived peptides block Akt-MADD phosphorylation by for example, binding to a kinase active site of Akt or through steric hindrance to phosphorylation. Suitable lengths of MADD-derived peptides or polypeptides include for example, 25, 50, 75, 100, 200, 300, 400, 500, 600, 750, 1000, or full-length MADD with one or more mutations that render the MADD incapable of binding to DR. In some aspects amino acid sequences comprising contiguous sequences from MADD e.g., amino acid positions 70-1041, 50-1100, 173-1041, 70-173, 50-200, 150-1100, 1-173, 1-1041 and 1-200 of MADD are suitable such that interact with Akt to prevent Akt phosphorylation of MADD and thereby preventing pro-survival functions of MADD in a cancer cell.
In certain embodiments, the compositions disclosed herein e.g., nucleic acid molecules that selectively down-regulate MADD expression of peptide molecules that block Akt-phosphorylation of MADD may be administered as an adjuvant therapy along with chemotherapy or radiation therapy. The compositions may also be administered prior to a standard cancer therapy and may optionally be administered along with or after cancer therapy. Examples of cancer therapy include for example, doxorubicin, cisplatin, antibody-therapy, and radiation therapy. In some aspects, the compositions disclosed herein sensitize resistant cancer cells, e.g., wherein the cancer cell is resistant to TRAIL-induced apoptosis.
A method of identifying an inhibitor of the phosphorylation of MADD by Akt includes:
Suitable inhibitors include for example, small molecules and peptides. Antibody for identifying an inhibitor is generated against a peptide epitope selected from the group e.g., CRQRRMpSLRDDTS (SEQ ID NO: 7) (S-70), GSRSRNSpTLTSL (SEQ ID NO: 8) (T-173), and KRKRSPpTESVNTP (SEQ ID NO: 9) (T-1041), wherein Mp or Sp or Pp is a phosphorylated amino acid. In an embodiment, the testing is performed in the presence of phosphorylated MADD.
Suitable assays to identify inhibitors of MADD phosphorylation include for example, a high throughput screening system for agents that can bind to MADD and prevent its prosurvival function and thus result in enhanced apoptosis. In an aspect, the screening method may include two steps. For example, first to screen for the compound that can enhance apoptosis and second to determine the compound's binding to phosphorylated MADD. Alternatively, screening for compounds that can bind only to phospho MADD followed by testing to determine the compounds' abilities to induce/enhance apoptosis. Standard assays techniques are used to determine the binding efficiency to the phosphopeptides of MADD.
In another aspect, in vitro assays are designed to monitor/identify the ability of the inhibitors to block either MAPK's or Akt's ability to phosphorylate MADD by analyzing the binding of anti-phospho-MADD antibodies. If, in the presence of a candidate agent (e.g., inhibitor), the phosphorylation of MADD does not account at one or more of the positions at 70, 173 or 1041 as compared to the control, as determined by e.g., lack of detection by the phospho antibodies, then the candidate agent is further evaluated for its ability to induce apoptosis in a cancer cell.
Phosphor peptides derived from MADD are used in NMR based screening for compounds. For example, each of these peptides has a particular NMR spectrum that may change upon binding of a small molecule or a compound. Based on the alterations in the NMR spectrum upong being exposed or contacted with a candidate agent (e.g., small molecule), a large number of candidate agents for example, from a commercially available library (e.g., SPECS) or any other collection, is screened in a high-through put fashion to identify agents that selectively bind or interact with the phosphorylated MADD or a peptide thereof.
A purified mutant MADD polypeptide includes one more mutations at amino acid positions 70, 173, and 1041. In an embodiment, the MADD polypeptide includes an amino acid sequence of SEQ ID NO: 3 that has amino acids at positions 70, 173, and 1041, which are all mutated such that Akt does not phosphorylate MADD.
To determine the requirement of DENN-SV in cancer cell growth and proliferation, exon 13L- and 16-specific shRNAs were expressed in HeLa cells. Significant increase in spontaneous apoptosis was observed when IG20pa/MADD, but not when IG20pa/IG20-SV2, were down-modulated (
Cancer cells die as a consequence of apoptosis due to prolonged arrest in either G1/S or G2/M phases of cell cycle or due to their inability to replicate. Diminished viability of cells upon Mid- and 13L-shRNA expression (
Apoptosis was consistently higher in 13L-treated, relative to Mid-treated, cells (
Unlike HeLa cells that express all 4 IG20-SVs, the PA-1 (ovarian carcinoma) cell line expresses predominantly MADD and DENN-SV. This cell line was used to unambiguously demonstrate the role of MADD in promoting cancer cell survival. The results obtained on cell proliferation and cell cycle progression with PA-1 cells were very similar to the observations made in HeLa cells, and thereby supported the finding that MADD but not any of the other three IG20-SVs can promote cancer cell survival.
Down-modulation of MADD alone can cause spontaneous apoptosis. However, over-expression of exogenous MADD had no discernible effect on induced apoptosis or cell proliferation. Although the mode of action of MADD is not known, it can bind to death receptors (DRs) and thus might prevent spontaneous oligomerization of DRs that leads to apoptosis. If the endogenous MADD (a pro-survival molecule) was sufficient to prevent DR oligomerization, expression of exogenous MADD might have had little or no effect. On the other hand, either down-modulation of MADD or expression of exogenous IG20pa (a pro-apoptotic molecule), which might act as a dominant negative, renders cells susceptible to apoptosis by facilitating DR oligomerization. In contrast, expression of exogenous DENN-SV (an anti-apoptotic molecule) stabilizes or synergizes MADD and prevent apoptosis. While IG20pa enhanced TRAIL-induced apoptosis was accompanied by increased recruitment of death-inducing signaling complex (DISC) and caspase activation; DENN-SV induced resistance was characterized by enhanced NFkB activation. Data presented herein demonstrate the requirement of MADD for cancer cell survival and the clinical implication of selective abrogation of MADD.
Data presented herein also demonstrated that MADD abrogation can lead not only to spontaneous apoptosis but also to enhanced TRAIL-induced apoptosis resulting from caspase-8 activation at the DRs and strongly indicated that MADD acts as a negative regulator of caspase-8 activation in cancer cells.
The levels of expression of DRs and DcRs, or their ligands were unperturbed. Expression of CrmA, a known inhibitor of caspase-1 and -8, or DN-FADD that competes with endogenous FADD conferred resistance to spontaneous apoptosis. Increased activation of caspase-8 at the DISC was evident from an increase in the p43/p41 fragments. Caspase-8 activation resulting from MADD abrogation was not accompanied by an increase in the recruitment of FADD or caspase-8 to the DISC (
Although MADD transcripts are depleted by 24 h post-transduction of shRNAs, it takes 72 h for spontaneous apoptosis to set in (
Over-expression studies failed to indicate a role for MADD in enhanced cell survival. This would suggest that endogenous MADD might be sufficient to fully exert its function and the effects of exogenous MADD, if any, thus may not be apparent. Similarly, although exogenous IG20pa can enhance induced-apoptosis, IG20pa knock-down failed to confer resistance to TRAIL-induced apoptosis. Since IG20pa can be a part of TRAIL-induced DISC, it may act as a dominant-negative MADD and render cells more susceptible to induced-apoptosis. Similarly, over-expression of DENN-SV enhanced cell proliferation and resistance to apoptosis and expression of DENN-SV in the absence of MADD did not prevent apoptosis. DENN-SV, due to its ability to enhance NFκB-activation, might complement the pro-survival function of MADD and thus, upon over expression, can enhance cell survival and proliferation.
Although the mode of action of MADD is not yet known, it can bind to DRs, but not to FADD or caspase-8, and prevent activation of caspase-8 without affecting DR-FADD or FADD-caspase-8 interactions (
Breast cancer is a leading cause of cancer related deaths among women. Current therapies, such as paclitaxel, can be toxic and may result in severe side effects including hypotension, bradycardia and peripheral neuropathies. Therefore, new safer treatments are desirable. The IG20 gene plays a critical role in cell proliferation and apoptosis and is highly over-expressed in human tumors and cancer cell lines. It encodes four splice variants (SVs) IG20pa, MADD, IG20-SV2 and DENN-SV. Earlier, over-expression studies showed that IG20pa can act as a tumor suppressor, while DENN-SV can act as an oncogene. More recently, selective knock-down of IG20 SVs revealed that the MADD isoform alone is necessary and sufficient for HeLa and PA-1 cell survival. Knock-down approach involves using a GFP-tagged lentivirus to deliver siRNAs into cells and measuring apoptosis by TMRM staining followed by flow cytometry. Interestingly, normal cells are relatively unaffected by the down-modulation of MADD. MADD protects these cancer cells by acting as a negative regulator of caspase-8 activation and therefore, suggests that MAD knock-down might synergize therapies for breast cancer. Knock-down of MADD, in MCF-7 human breast cancer cell line, and not other isoforms resulted in enhanced spontaneous apoptosis compared to cells treated with a scrambled siRNA. Moreover, knock-down of MADD in MCF-7 cells followed by treatment with sub-optimal doses of TRAIL resulted in enhanced cell death compared to either treatment alone. These data support the notion that knock-down of MADD may synergize with different modalities of breast cancer therapy resulting in reduced side effects and improved quality of life.
The MADD cDNA sequence is available on the GenBank database using accession number: NM—130470. The interfering RNAs disclosed herein that down-regulate MADD are intended to target any allelic variants and naturally occurring mutants of MADD, and polymorphisms that occur in MADD that are found in a particular segment of the population. In other words, sequences that are highly similar (e.g., about 95% at the amino acid level and about 75% at the nucleic acid level) that represent naturally occurring variations in MADD are within the scope of the disclosure, wherein the shRNAs and siRNAs disclosed herein are capable of down-regulating the expression of such sequences. The shRNA sequences presented herein provide a framework and the specification provides guidance to design additional nucleic acid sequences capable of producing interfering RNAs to down-regulate MADD splice variant. MADD sequences that are about 80% or 90% or 95% similar at the nucleic acid level to the MADD sequence disclosed herein are also down-regulated. Accordingly, nucleic acid sequences that generate shRNAs can be redesigned to accommodate the variations if those variations occur within the target region.
Data presented herein demonstrate that MADD has a dual apoptosis-regulating function depending on its phosphorylation status. It prevents apoptosis when it is phosphorylated by Akt, but when non-phosphorylated, it triggers apoptosis. Furthermore, phosphorylated MADD quenches the extrinsic apoptotic pathway, while nonphosphorylated MADD triggers apoptosis by releasing the extrinsic pathway on hand, and activating the intrinsic pathway on the other. Therefore, phosphorylation-dependent dual functions of MADD play an important role in cellular homeostasis. MADD has three potential Akt phosphorylation sites, serine-70, threonine-173 and threonine-1041. Data demonstrate that Akt can phosphorylate MADD at these three sites. And only phosphorylated MADD binds to DR4 and inhibits both spontaneous and TRAIL-induced activation of the extrinsic apoptotic pathway. This binding is significantly weak when any of the three phosphorylation sites is mutated. This indicates that phosphorylation of all three sites contributes to MADD binding to DRs. Non-phosphorylated MADD neither binds to DR nor can prevent activation of the extrinsic apoptotic pathway. In contrast, non-phosphorylated MADD now binds to the protein 14-3-3 and dislodges Bax from 14-3-3 resulting in Bax translocation to mitochondria leading to the activation of the intrinsic apoptotic pathway. Furthermore, TRAIL-induced apoptosis was dependent upon reduced levels of Akt and MADD phosphorylation. Thus, Akt phosphorylated MADD, contributes to cell survival, while the non-phosphorylated MADD allows activation of extrinsic and intrinsic apoptotic pathways leading to cell death.
MADD has a dual function in regulating apoptosis depending on its phosphorylation by Akt. Akt can phosphorylate the proapoptotic protein Bad and facilitates its binding to 14-3-3 in the cytosol and prevents translocation to mitochondria where it can exert its proapoptotic effect. Bax is also regulated by Akt signaling pathway in that it phosphorylates Bax on Ser184 and prevents its translocation to mitochondria. In contrast, phosphorylation of MADD by Akt not only allows it to bind to DR and block activation of the extrinsic pathway, but it also prevents its interaction with 14-3-3 which is required for Bax release leading to activation of the intrinsic pathway. Thus, MADD can be either anti-apoptotic or pro-apoptotic depending on its phosphorylation by Akt.
The extrinsic apoptotic pathway is quenched by MADD upon phosphorylation. MADD may exert its anti-apoptotic effect by binding to DR4, and preventing death receptor oligomerization. Present data show that only phosphorylated MADD can bind to DR4 and attenuate TRAIL-induced DISC formation. It is possible that phosphorylation facilitates MADD association with DR4 and/or controls MADD cellular localization.
The intrinsic apoptotic pathway is provoked by the non-phosphorylated MADD Mitochondria play a pivotal role in apoptosis by releasing various apoptotic molecules into the cytoplasm triggered by the translocation of proapoptotic proteins into mitochondria. For example, Bax resides predominantly in the cytoplasm of healthy cells, but upon treatment with apoptotic stimuli it translocates to mitochondria where it forms oligomers and causes cytochrome c release. Akt can suppress Bax translocation to mitochondria by facilitating its interaction with 14-3-3 in the cytosol. Other proapoptotic proteins, such as hepatitis C virus core protein, can interact with 14-3-3 thereby allowing Bax to dissociate from 14-3-3 and translocate to mitochondria leading to apoptosis. Similarly, non-phosphorylated form of MADD can interact with 14-3-3 causing release of Bax from Bax/14-3-3 complex and its translocation to mitochondria. Since 14-3-3 can also bind to other proapoptotic proteins (e.g. Bad), it is possible that MADD can similarly disrupt those associations.
Although only phosphorylated forms of some apoptotic proteins such as Bad and FOXO3a can bind to 14-3-3, the findings presented here are consistent data that show protein binding to 14-3-3 that is not necessarily dependent on phosphorylation.
How can nonphosphorylated MADD influence the association of 14-3-3 with Bax? c-Jun NH2-terminal kinase (JNK) promotes Bax translocation to mitochondria through phosphorylation of 14-3-3. Phosphorylation of 14-3-3 leads to Bax dissociation and translocation to mitochondria. A C-terminal fragment of MADD (amino acids 1396-1588) without the three phosphorylation sites is able to activate JNK, whereas the intact MADD is unable to activate JNK. This may explain why only the non-phosphorylated MADD can dissociate Bax from 14-3-3.
TRAIL initiates apoptosis by reducing MADD phosphorylation levels. TRAIL is able to induce apoptosis in a wide variety of cancer cells with minimal cytotoxity to most normal cells and tissues, and thus considered a promising cancer therapy. Resistance to TRAIL-mediated apoptosis induction in cancer cells exists. Data presented herein show that TRAIL could not only reduce Akt activity but also the levels of MADD phosphorylation, and indicates that MADD can act as a key regulator of TRAIL induced apoptosis through Akt phosphorylation.
The association of phosphorylated MADD with DR4 can quench the TRAIL-induced death signal mediated through the extrinsic pathway. However, upon TRAIL treatment the levels of nonphosphorylated MADD increases resulting in its dissociation from DR4, which allows for the activation of the extrinsic apoptotic pathway. TRAIL can also initiate the intrinsic pathway by increasing the levels of non-phosphorylated MADD, which upon binding to 14-3-3 causes Bax release and translocation to mitochondria. Thus MADD can be a downstream target of TRAIL that facilitates TRAIL-induced activation of the intrinsic pathway.
The pathophysiological significance of MADD phosphorylation by Akt. One of the major obstacles for effective cancer therapy is the development of resistance to chemotherapy and radiation therapy. The molecular mechanisms by which the resistance develops appear different for different cancers. Activation of Akt is common in many cancers, and promotes cell survival. Akt is activated in multiple myeloma, lung cancer, breast cancer, brain cancer, gastric cancer, acute myelogenous leukemia, endometrial cancer, melanoma, renal cell carcinoma, colon cancer, ovarian cancer, and prostate cancer and may contribute to less than optimal outcome following radiation and chemotherapy resulting in poor prognosis. Thus, targeting PI3K and Akt is an effective adjuvant therapy for cancers. Similarly, TRAIL may be an effective treatment for a variety of cancers. However, the possibility that the treatment itself can quickly induce resistance and promote metastasis can limit its potential usefulness. Therefore, by blocking PI3K or Akt, or modulating Akt substrates, resistance may be minimized and could serve as effective adjuvant therapies. The results provided herein indicate that modulating the Akt-MADD signaling pathway represents an effective adjuvant therapeutic approach to treat cancer.
MADD-SV acts as a negative regulator of caspase-8 activation and promotes cancer cell survival and prevents cell apoptosis. The effect of MADD-SV on caspase-8 activation is not due to the direct association of MADD-SV with caspase-8. How MADD-SV of the IG20 gene acts a negative regulator of caspase-8 activation is analyzed herein.
One possible mechanism is that binding of MADD-SV to the receptor results in the activation of a prosurvival pathway which can antagonize caspase-8 activation. It was tested whether NF-kB pathway, which is a well characterized survival pathway, is involved in negative regulation of caspase-8 activation by MADD-SV. Since over-expression of MADD-SV did not have any role in the NF-kB pathway, this possibility was tested using the knock-down approach employing TNF-α stimulation. Inability to activate NF-kB pathway can render cells extremely susceptible to apoptosis. Data presented in
Whether the activation of MAP kinase pathway which is another important survival pathway, plays a role in antagonizing caspase-8 activation by MADD-SV was examined. Over-expression studies have shown that MADD-SV binds to TNFR1 and can activate ERK1/2 MAP kinases. It was determined whether endogenous MADD-SV plays an essential role in TNF-α induced ERK activation pathway thereby antagonizing caspase-8 activation. This phenomenon was studied in a physiologically more relevant system by using ShRNA to knock-down endogenous isoforms of the IG20 gene including MADD-SV.
The four IG20-SVs (IG20pa, MADD-SV, IG20-SV2 and DENN-SV) show a very high degree of sequence homology and can only be differentiated by the differential splicing of exons 13L (130 base pairs) and 16E (60 base pairs). Hence delineating the roles of individual isoforms of IG20 is often difficult. This intricate task of identifying which IG20 isoform is critical for TNF-α induced ERK activation was undertaken by taking advantage of the differential splicing of exons 13L and 16E and the post-transcriptional sequence-specific mode of action of ShRNA which can down-modulate all or specific combination of IG20 isoforms. The knock-down of all IG20-SVs (
The effects of down-modulation of IG20-SVs esp. MADD-SV were analyzed on the phosphorylation of the down-stream substrate of ERK like p90RSK. The kinase, p90RSK upon phosphorylation by ERK1/2, with which it is physically associated in the cytoplasm, translocates to the nucleus and activates transcriptional factors like CREB and c-FOS which play a critical role in cancer cell growth, differentiation and development. The down-modulation of MADD-SV has resulted in near complete absence of p90RSK phosphorylation (
Two major problems of cancer chemotherapy are toxicity to normal cells and failure to kill cancer cells. The damage is caused to both normal and cancer cells, and this damage is then passed through multiple steps into cell death, likely through activation of caspases and apoptosis. When these steps are compromised by antiapoptotic events, such as p53 mutation or overexpression of Bcl-2, the therapy fails. An alternative strategy is to design a treatment that activation caspases directly by activating the death receptor complexes, resulting in activation of their corresponding initiator caspases like caspase-8. Down-modulation of MADD-SV of the IG20 gene provides a tool for molecular targeted therapies. First, down-modulation of MADD-SV does not after the growth of normal cells since it is a protein over-expressed in cancer cells and plays a prominent role only in cancer cell growth. Second, down-modulation of endogenous MADD-SV abrogates ERK1/2 phosphorylation and prevents the cancer growth mediated by TNF-α induced ERK MAP kinase activation. Third, down-modulation of MADD-SV alone is sufficient to directly activate caspase-8 and subsequently the effector caspase-3 upon TNF-α stimulation and thereby promote apoptosis of cancer cells. Down-modulation of endogenous MADD-SV works like a double-edged weapon in preventing cancer growth mediated by TNF-α induced ERK activation. In one way it abrogates pro-survival ERK activation and in other way it activates caspases and brings about apoptosis.
Specific down-regulation of MADD splice variant or inhibition of its activity is also possible by agents such as a small molecule, a peptide nucleic acid, an antibody, inhibitor compound, and synthetic nucleic acids that specifically bind to RNA. RNA/DNA hybrids and down-regulate MADD isoform expression.
Nucleic acid or nucleic acid sequence or polynucleotide or polynucleotide sequence refers to the sequence of a single- or double-stranded DNA or RNA molecule of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide or ribonucleotide bases, respectively.
An isolated nucleic acid sequence is substantially separated or purified from other nucleic acid sequences with which the nucleic acid is normally associated in the cell of the organism in which the nucleic acid naturally occurs. The term includes nucleic acids that are biochemically purified so as to substantially remove contaminating nucleic acids and other cellular components. The term also includes recombinant nucleic acids and chemically synthesized nucleic acids. The term “substantially purified”, as used herein, refers to a molecule separated from other molecules normally associated with it in its native state. For example, a substantially purified molecule is the predominant species present in a preparation, such as, isolated BTI nucleic acid after a PCR. In one embodiment, a siRNA molecule includes a double stranded RNA wherein one strand of the RNA is complimentary to the RNA of MADD splice variant. In another embodiment, a siRNA molecule of the invention includes a double stranded RNA wherein one strand of the RNA comprises a portion of a sequence of RNA having MADD or IG20 gene splice variant sequence. In another embodiment, a siRNA molecule includes a double stranded RNA wherein both strands of RNA are connected by a non-nucleotide linker. Alternately, a siRNA molecule includes a double stranded RNA wherein both strands of RNA are connected by a nucleotide linker, such as a loop or stem loop structure.
Short hairpin RNA (shRNA) contains complementary sense and antisense sequences of a target gene linked by a loop structure. The target sequence starts as a dsDNA cloned into an expression vector that is transcribed to form an shRNA. In the cytoplasm, shRNA is cleaved by Dicer generating short sequences of RNA that can inhibit gene expression by RNAi. The benefit to shRNA is stable transfection of cell lines and enables a single gene in each cell to be targeted. To construct shRNA vectors, see McIntyre and Fanning (2006), Design and cloning strategies for constructing shRNA expression vectors, BMC Biotechnol. 2006; 6: 1, incorporated herein by reference in its entirety.
In one embodiment, a single strand component of a siRNA molecule is from about 14 to about 50 nucleotides in length. In another embodiment, a single strand component of a siRNA molecule is about 15-20, 15-21, 14-25, 16-30 or 20-25 nucleotides in length. In yet another embodiment, a single strand component of a siRNA molecule is about 23 nucleotides in length. In one embodiment, a siRNA molecule is from about 20 to about 40 nucleotides in length.
In an embodiment, an antisense nucleic acid molecule, decoy RNA, dsRNA, siRNA, shRNA, or aptamer, nucleic acids include at least one nucleic acid base modification.
In another embodiment, an antisense nucleic acid molecule, decoy RNA, dsRNA, siRNA, shRNA, or aptamer, nucleic acids of the invention comprises at least one phosphate backbone modification.
Methods for treatment of cancer are described wherein cancer includes for example breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer, comprising administering to a subject, a nucleic acid molecule or antisense nucleic acid molecule or other nucleic acid molecule capable of down-regulating MADD expression or other splice variant of IG20 gene under conditions suitable for said treatment. Any cancer cell that expresses one or more of the IG20 splice variants including MADD is suitable for cancer therapeutics using the compositions disclosed herein.
In another embodiment, conventional or other known drug therapies to be used along with the down-regulation of MADD or prior to or after MADD down-regulation include monoclonal antibodies, specific inhibitors, chemotherapy, or radiation therapy or a combination thereof for cancer.
Specific chemotherapy include paclitaxel, docetaxel, cisplatin, methotrexate, cyclophosphamide, 5-fluoro uridine, Leucovorin, Irinotecan (CAMPTOSAR™ or CPT-11 or Camptothecin-11 or Campto), Paclitaxel, Carboplatin, doxorubicin, fluorouracil carboplatin, edatrexate, gemcitabine, or vinorelbine or a combination thereof can be administered along with agents capable of down-regulating MADD or prior to or after down-regulation of MADD.
Antisense nucleic acid refers to a nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (peptide nucleic acid) interactions and alters the activity of the target RNA. Typically, antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop. Thus, the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both. In addition, antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
Double stranded RNA or dsRNA refers to a double stranded RNA that matches a predetermined gene sequence that is capable of activating cellular enzymes that degrade the corresponding messenger RNA transcripts of the gene. These dsRNAs are referred to as short intervening RNA (siRNA) and can be used to inhibit gene expression. The term “double stranded RNA” or “dsRNA” as used herein refers to a double stranded RNA molecule capable of RNA interference “RNAi”, including short interfering RNA “siRNA”.
Splice variant refers to a specific isoform of IG20 gene that is expressed in one or more cell type by alternate splicing.
Consisting essentially of means that the nucleic acid molecule includes a nucleic acid sequence capable of down-regulating a specific splice variant of IG20 gene. Other sequences can be present which do not interfere with the activity.
In another aspect nucleic acid molecules or antisense molecules that interact with target RNA molecules and down-regulate MADD activity are expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors are preferably DNA plasmids or viral vectors. Enzymatic nucleic acid molecule or antisense expressing viral vectors can be constructed based on adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the nucleic acid molecules are delivered as described herein, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of shRNA/siRNA/anti-sense nucleic acid molecules. Such vectors can be repeatedly administered as necessary. After being expressed, the interfering nucleic acid molecules bind to the target RNA and down-regulate its function or expression. Delivery of nucleic acid molecule or antisense expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells explanted from the patient or subject followed by reintroduction into the patient or subject, or by any other means that would allow for introduction into the desired target cell. Antisense DNA can also be expressed via the use of a single stranded DNA intracellular expression vector.
In addition, the term “pharmaceutically effective amount” or “therapeutically effective amount” refers to an amount (dose) effective in reducing tumor growth or tumor size or cancer metastasis of a patient, having, for example, cancer. It is also to be understood herein that a “pharmaceutically effective amount” may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents. In the case of the present disclosure that relates to down-regulation of IG20 splice variants, a “pharmaceutically effective amount” may be understood as an amount of shRNA or siRNA that specifically down-regulates one or more of the IG20 splice variants including MADD which may for example, suppress (e.g., totally or partially) the expression of IG20 splice variants. The dose may be of any suitable form.
Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar to the art, including, but not limited to, encapsulation in liposomes, by iontophoresis, or by an incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Other approaches include the use of various transport and carrier systems, for example, through the use of conjugates and biodegradable polymers.
The molecules disclosed herein can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state in a subject.
The negatively charged polynucleotides can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. The compositions can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the other compositions known in the art.
Pharmaceutically acceptable formulations of the compounds are described. These formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.
Systemic administration refers to in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue, e.g., tumor tissue. The use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types. A liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.
The nucleic acid molecules disclosed herein and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule and a pharmaceutically acceptable carrier. One or more nucleic acid molecules can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Pharmaceutical compositions that consist essentially of the nucleic acid sequences to knock-down a specific IG20 splice variant may include about 0.1 μg to 1 mg of nucleic acid per kg of body weight. Variations in dosage and effects can be optimized using skills known to a skilled artisan.
In another aspect nucleic acid molecules include an expression vector that includes nucleic acid sequence encoding at least one of the nucleic acid molecules, in a manner which allows expression of that nucleic acid molecule. The expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein the sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
The following examples are to be considered as exemplary and not restrictive or limiting in character and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.
In order to determine which of the IG20-SVs contribute to apoptosis and cell proliferation, siRNA (small inhibitory RNA) approach was used to selectively knockdown IG20-SVs (Table 1). The siRNAs were cloned into the pSUPER vector to allow for expression of shRNAs (small hairpin RNAs). An aspect of such a vector is shown in
The 13L- or 16E-shRNAs were highly specific and had little or no effect on IG20-SVs devoid of the targeted exon. To further confirm specificity of Mid-shRNA, silent mutations were created in cDNAs encoding IG20-SVs at sites corresponding to the 5th, 7th, 11th and 14th nucleotides of the Mid-shRNA. These mutations neither affected the amino acid sequence nor protein expression. Mutant-IG20-SV constructs were cotransfected with the pSUPER-shRNA and assessed for their expression. Mid-shRNA failed to down-modulate the mutant-proteins (
The shRNAs described herein were cloned into a self-inactivating lentivirus vector (pNL-SINGFP) and generated SUP (vector control), Mid, 13L, 16E and SCR (negative control shRNA) constructs to target specific combinations of endogenous IG20-SVs. An illustration of such a vector is shown in
Differential down-modulation of endogenous IG20-SVs in HeLa cells was analyzed. 1 μg total RNA obtained from HeLa cells at 72 hours post-transduction was used for RT-PCR. The products were separated on a 2% agarose gel. Amplification of all four IG20-SVs using F2-B2 primers was performed. Amplification of IG20pa and MADD using 13L-Forward and B2-Reverse primers was performed. The transduction efficiency was over 80% as determined by GFP expression. Relative to controls (SUP and SCR), HeLa cells expressing Mid-shRNA showed decrease in all IG20-SVs. While, 13L-shRNA caused near complete abrogation of only IG20pa and MADD, 16E-shRNA decreased expression of only IG20pa and IG20-SV2, without affecting the expression of the other variants. This was confirmed using 13L-forward and B2-reverse primers, and F2-forward and 16E-reverse primers, that amplify only IG20pa and MADD, and not DENN-SV. The effects of loss of various endogenous IG20-SV transcripts on the ability of cancer cells to survive were investigated.
Spontaneous cell death was determined by nuclear condensation (Hoechst 33342) and loss of mitochondrial membrane potential (TMRM staining) (
Using a pan-caspase inhibitor, a general increase in caspase activation was observed in cells that had reduced MADD expression (
In order to assess the effects on cell growth and proliferation, various shRNA-expressing viable cells were counted. Relative to controls, a significant decrease in the numbers of viable cells expressing Mid- and 13L-shRNA was observed (
In HeLa cells, down-modulation of all four, or a combination of IG20pa/MADD or IG20pa/IG20-SV2 variants was demonstrated. To unequivocally demonstrate that MADD is required for cell survival, PA-1 ovarian carcinoma cells were used that essentially express only MADD and DENN-SV and thereby facilitate exclusive down-modulation of MADD. The effects of IG20 down-modulation in PA-1 cells were analyzed. Down-modulation of endogenous IG20 in PA-1 cells was analyzed. RT-PCR of IG20-SVs using F2-B2 primers from PA-1 cells 72 hours post-transduction was performed. Amplification of only IG20pa and MADD using 13L-F and B2 primers was performed. Nuclear Condensation was also determined. 72 hours post-transduction, PA-1 cells were stained with Hoechst stain and analyzed by FACS. Mitochondrial Depolarization was performed. 72 hours post transduction, PA-1 cells were stained with TMRM and analyzed by FACS.
While treatment with Mid-shRNA down modulated both MADD and DENN-SV, treatment with 13L-shRNA abrogated only MADD expression. Further confirmation was obtained by using a different set of primers to amplify MADD and IG20pa. Seventy two hours post-transduction with 13L-shRNA 50% of the cells underwent spontaneous apoptosis.
The ability of cells to proliferate was also determined. Equal numbers of PA-1 cells (24 hr post-transduction) were cultured and the number of viable cells determined at various time points (
Down modulation of IG20 transcripts upon treatment of cells with lentiviruses expressing different shRNAs was monitored by RT-PCR. The IC20 transcripts were significantly down-modulated by 24 h (
Although the IG20 transcripts are significantly down-modulated by 24 hrs, the cells do not undergo spontaneous apoptosis until 72 hours post shRNA induction. This is most likely due to the time required for complete degradation of the remaining endogenous proteins. Therefore, at thirty-six hours post-shRNA transduction when there is no indication of spontaneous apoptosis, HeLa cells were treated with various concentrations of TRAIL for different durations and assayed for apoptosis. Cells devoid of MADD showed enhanced TRAIL-induced apoptosis as indicated by significant increases in caspase-3 activation (
Since increases in the levels of expression of DRs and their ligands, or decreases in DcRs can result in oligomerization of death receptors followed by apoptotic cell death, the levels of their expression in HeLa and PA-1 cells were tested. No significant difference in the levels of expression in various sh-RNA transduced cells relative to control cells was observed, and indicated that spontaneous apoptosis resulting from MADD down-modulation was not due to perturbations in the levels of DR4, DR5, Fas, FasL, TRAIL, DcR1 and DcR2 expression on the cell surface. Surface expression of DRs, DcRs, and their ligands are not altered upon MADD abrogation. HeLa cells transduced with the indicated shRNAs for 48 h were collected in enzyme-free cell dissociation buffer and stained with antibodies (specific to DR4, DR5, DcR1, DcR2, TRAIL, Fas and FasL or isotype controls) conjugated to PE.
Only MADD-depleted HeLa and PA-1 cells (Mid and 13L cells) showed higher amounts of the intermediate active-caspase-8 proteins (p43/41). To gain insight into the mechanism underlying MADD-mediated cell survival, whether caspases were activated upon abrogation of MADD expression examined. HeLa cells and PA-1 cells were collected 48 h post-transduction and probed for caspase-8 and FADD, which are components of the DISC. As observed, the cleaved intermediate fragments of caspase-8 (early indication of caspase-8 activation) were detected only upon MADD abrogation, although the levels of pro-caspase-8, FADD, and actin were comparable in all cells. Only MADD-depleted HeLa and PA-1 cells (Mid and 13L cells) showed processing of pro-caspase-8 (p55/53), as indicated by the higher amounts of the intermediate active caspase-8 proteins (p43/41). Similarly, HeLa cells treated with 100 ng of TRAIL for different durations showed sustained caspase-8 activation and subsequent caspase-3 activation. TRAIL treatment results in recruitment of procaspase-8 to the receptors followed by its cleavage resulting in its activation. The cleaved intermediate fragments (p43/p41) and the catalytically active subunits of caspase-8 (p18) were detected upon TRAIL treatment. Caspase-8 is the initiator caspase that can cause activation of the effector caspase-3. A decrease in the amount of pro-caspase-3 indicates its cleavage to form active caspase-3 only in MADD-depleted cells. These results indicated that as early as 48 h after MADD abrogation, pro-caspase 8 (p55/53) is processed and cleaved into its intermediate (p43/41) active fragments. Similarly, HeLa cells treated with 100 ng of TRAIL for the indicated durations showed sustained caspase-8 activation and subsequent caspase-3 activation as evident from the increased levels of p43/p41 and p18 subunits of caspase-8, and a concomitant decrease in pro-caspase-3 levels only in MADD-depleted cells.
To more specifically analyze the DISC associated with TRAIL-bound death receptors, TRAIL-induced DISC was immunoprecipitated from HeLa cells using a biotinylated-antibody specific for TRAIL from HeLa cells that were either left untreated or treated with 250 ng of TRAIL for 30 min. Complexes were separated on 12% SDS-PAGE and subjected to immunoblotting with various antibodies that can specifically react with specific DISC components. Increased recruitment of FADD and pro-caspase-8 was observed after TRAIL treatment. Relative to control cells (SCR), TRAIL immunoprecipitates from MADD-depleted cells (13L and Mid) showed increased levels of intermediate fragments of caspase-8 (p43/41). However, increased caspase-8 activation (p43/41) was more apparent only upon MADD down-modulation.
Further evidence in support of caspase-8 activation in apoptosis induced by MADD abrogation was gained from experiments carried out with HeLa and PA-1 cell lines stably expressing DN-FADD (Dominant-negative FADD) or CrmA. The spontaneous cell death that occurs due to abrogation of endogenous MADD is dramatically inhibited by both CrmA and DN-FADD. These inhibitors of DISC formation also rendered MADD-depleted HeLa cells (36 hours post-ShRNA transduction) resistant to TRAIL-induced apoptosis (
Since caspase-8 plays an essential role in receptor-mediated apoptosis that is characterized by DISC formation, the status of DISC in cells undergoing apoptosis due to loss of endogenous MADD expression was examined. DR4 and DR5 from HeLa and PA-1 cells respectively were immunoprecipitated and probed for known DISC components. As caspase-8 plays an essential role in receptor-mediated apoptosis characterized by DISC formation, the status of DISC in cells undergoing apoptosis due to loss of endogenous MADD expression was examined. Forty-eight-hour transduced HeLa cells and PA-1 cells were collected and lysed. Lysates were normalized for protein concentration, and DR4s/DR5s were immunoprecipitated using specific antibodies. Separated immune complexes were immunoblotted using antibodies specific for caspase-8, FADD, and DR4/DR5. The caspase-8 and FADD and the DR4/DR5 immunoblots were exposed for 4 h and for 30 min, respectively. FADD and pro-caspase-8 were found to be associated constitutively with the DRs in cells with and without MADD abrogation. However, the intermediate cleaved fragments of caspase-8 (p43/p41) were detected only in MADD-depleted cells, which indicated that activation of caspase-8 at the DRs was associated with spontaneous apoptosis resulting from MADD abrogation.
FADD and pro-caspase-8 were found to be constitutively associated with the DRs in cells with and without MADD abrogation. However, the intermediate cleaved fragments of caspase-8 (p43/p41) were detected only in MADD-depleted cells, which suggested that activation of caspase-8 at the DRs was associated with spontaneous apoptosis.
TRAIL induced DISC was immunoprecipitated from HeLa cells using a TRAIL-specific antibody and subjected to SDS-PAGE followed by western blotting. Staining for various TRAIL-DISC components revealed co-precipitation of DR4, FADD and caspase-8. Relative to control cells (SCR), immunoprecipitates from MADD-depleted cells (13L and Mid) showed increased levels of intermediate fragments of caspase-8 (p43/41). This observation correlated with enhanced caspase-8 activity observed earlier in these cells upon TRAIL treatment (
To examine whether MADD confers resistance to apoptosis by interacting with caspase-8, MADD-YFP were expressed in HeLa cells and immunoprecipitated it using an IG20-specific antibody from lysates of cells that were left either untreated or treated with TRAIL (250 ng) for 30 minutes. These proteins were separated by SDS-PAGE and subjected to western blot analysis to probe for DISC components. DR4 but not caspase-8 co-precipitated with MADD in both untreated and TRAIL-treated cells. On the other hand, FADD co-precipitated with caspase-8 upon TRAIL treatment, while MADD and DR4 were not be co-precipitated with caspase-8 under either condition.
To determine whether MADD prevents caspase-8 activation by directly binding to caspase-8, HeLa cells were transfected with MADD-YFP. Thirty-six hours post-transduction, cells were either left untreated or treated with 250 ng of TRAIL for 30 min. Samples were lysed, and the lysates were normalized for protein concentration and pre-cleared. Equal amounts of proteins were incubated with either anti-IG20 or anti-caspase-8 antibody. Complexes were subjected to immunoblotting (WB, Western blot). Interestingly, endogenous DR4 but not caspase-8 co-precipitated with MADD in both untreated and TRAIL-treated cells. On the other hand, immunoprecipitation using a caspase-8-specific antibody followed by Western blotting showed co-precipitation of FADD with caspase-8 upon TRAIL treatment; whereas MADD and DR4 could not be co-precipitated with caspase-8 under either condition. The above results demonstrated that MAD D may exert its anti-apoptotic effect by directly binding to DR4 but not to caspase-8 or FADD.
To test whether MADD is a substrate of Akt, an in vitro assay was employed to test whether Akt could directly phosphorylate MADD. As shown in
To see whether phosphorylation is required for the prosurvival function of MADD, WT and mutant MADD were tested. Enforced expression of exogenous wtMADD alone could not induce cell death. Surprisingly, enforced expression of MADD70A, MADD173A or MADD1041A had minimal effects on cell death, MADD3A could induce a significant cell death (
MADD can bind to DR4 and thereby block the activation of the extrinsic apoptotic pathway. Results presented herein show that WtMADD can associate with DR4. While the association of DR4 with MADD single mutants was reduced, it was almost non-existant with MADD3A (
To explore the molecular mechanism by which nonphosphorylated MADD can trigger apoptosis, the cellular localization of phosphorylated and nonphosphorylated MADD was determined. Phosphorylated wtMADD is primarily located to the plasma membrane fraction, whereas nonphosphorylated MADD3A is located in the cytosolic fraction (
Since Bax can be sequestered by 14-3-3, it was tested whether the interaction of nonphosphorylated MADD with 14-3-3 could affect 14-3-3 and Bax interaction. Enforced expression of MADD3A, but not wtMADD, led to the disassociation of Bax from 14-3-3 (
To test whether Bax is the downstream mediator of apoptosis, Bax localization was analyzed. The Bax was translocated to the mitochondria in cells expressing MADD3A, but not wtMADD. Concomitantly, cytochrome c was released from the mitochondria into cytoplasm in cells expressing MADD3A (
DR4 is a TRAIL receptor and the inability of nonphosphorylated MADD to bind to DR4 led to consider whether MADD is a molecular target of TRAIL. As shown in
The ability of non-phosphorylated MADD to induce Bax translocation led to a consideration whether MADD is involved in the intrinsic apoptotic pathway initiated by TRAIL. MADD was redistributed from the plasma membrane to cytosol upon TRAIL treatment (
To gain insight into the time kinetics of TNF-α induced ERK phosphorylation, HeLa cells were treated with TNF-α (50 ng/mL) for different time periods. As shown in
The NF-κB-mediated survival pathway plays an important role in the resistance of cancer cells towards apoptosis. In order to test the possibility that down-modulation of MADD-SV negatively affects NF-κB activation, the effect of TNF-α treatment in shRNA transduced cells was determined. HeLa cells were treated thirty-six hours post-transduction with 50 ng of TNF-α and collected them as indicated in
In order to test whether IG20 gene is involved in the activation of other MAP kinases like JNK and p38, HeLa cells transduced with different ShRNA expressing lentiviruses were probed for P-JNK1/2 and P-p38. It was shown in
All the splice variants of the IG20 gene have a C-terminal DDHR (death domain homology region) with which they interact with death domain containing receptors like TNFR1. This interaction probably facilitated IG20 to play a prominent role in TNF-α induced ERK activation. The other major ERK activation pathway relevant to cancer is by growth factors binding to receptor tyrosine kinases. The results presented in
Whether MADD isoform alone is playing a critical role in TNF-α induced ERK activation was determined. Lentiviruses expressing different ShRNA can knockdown all or a select combination of IG20-SVs but not individual isoforms alone. Selective knockdown of MADD-SV and IG20pa (using 13L ShRNA expressing lentivirus) could significantly reduce ERK1/2 activation. To determine the roles of IG20pa-SV and MADD-SV in TNF-α induced ERK activation, 16E ShRNA expressing lentivirus which knocks out IG20pa-SV and IG20-SV2 but spares MADD-SV along with DENN-SV was used. The results presented in
The effect of down-modulation of IG20-SVs on the phosphorylation of a downstream substrate of ERK and an important kinase, p90RSK was investigated. Down-modulation of IG20-SVs employing Mid ShRNA has led to a dramatic decrease in phosphorylation of p90RSK (
Design of siRNA. The siRNAs used to target all, or a combination of IG20-SVs, are shown in Table I. Several putative siRNA sequences to target IG20 transcripts were obtained from the Dharmacon (Lafayette, Colo.). The most suitable sequences were sorted out based on less than 50% GC content, high AU content towards the 3′ end and no inverted repeats within the siRNA region (Reynolds et al. (2004), Nat Biotechnol, 22(3), 326-30.).
Plasmid Construction. The siRNAs were cloned into the pSUPER vector using BglII and HindIII sites (Brummelkamp et al., (2002), Science, 296(5567), 550-3.) to generate pS-Mid, pS-13L, pS-16E and pS-SCR plasmids. The respective shRNA cassettes (including the H1 RNA promoter and the shRNA) were excised form the pSUPER plasmids using XbaI and Clal sites and ligated into the pNLSIN-CMV-GFP vector (Lee et al., (2003), J Virol. (22), 11964-72.) to generate SUP (vector control), Mid, 13L, 16E and SCR (negative control) lentivirus constructs. The pcTat, pcRev and pHIT/G were gifts from Dr. B R Cullen and Dr. T J Hope. The IG20pa-YFP plasmid (Ramaswamy et al., (2004), Oncogene, 23(36), 6083-94) was used as a backbone to sub-clone MADD-YFP and DENN-SV-YFP plasmids from respective pBKRSV plasmids (Al-Zoubi et al., (2001), J Biol Chem, 276 (50), 47202-11.) using MluI and EcoRV sites. The DENN-SV-YFP-mut, MADD-YFP-mut and IG20-YFP-mut constructs were generated using the Quickchange XL site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) according to the manufacturer's protocol. Briefly, the primers 5′CGGAACCACAGTACAAGCTTTAGCCTCTCAAACCTCACACTGCC3′ (SEQ ID NO: 13) (forward) and 5′GGCAGTGTGAGGTTTGAGAGGCTAAAGCTTGTACTGTGGTTCCG3′ (SEQ ID NO: 14) (reverse) were designed to insert silent mutations at four sites in the cDNAs without affecting the amino acid sequence, as shown in bold, and were used to amplify the mutant-YFP-cDNAs. HindIII site in the mutants, generated due to base substitutions, was used to identify positive clones which were subsequently confirmed by sequencing.
Cell Culture. 293T cells, HeLa cells and PA-1 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, CA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin-G and 100 μg/mL streptomycin. All cell lines were maintained at 37° C. in a humidified chamber with 5.5% CO2.
Screening of shRNAs. IG20-YFP constructs were co-transfected with different pSUPER-shRNA constructs in different ratios (1:1, 1:3, 1:7) into 293T cells using Calcium phosphate. 24 hours post-transduction, cells were trypsinized, collected and washed twice in cold PBS, and analyzed for YFP expression by flow cytometry using a FACS Calibur (Becton Dickinson, N.J.).
Lentivirus production. Sub-confluent 293T cells grown in 100 mm plates were co-transfected with 10.8 μg of the respective lentivirus vector, 0.6 μg pcRev, 0.6 μg of pcTat and 0.3 μg of pHIT/G using calcium phosphate. Culture medium was replaced 16 hours later, and the supernatant was harvested 40 hours post-transfection and filtered using a 0.45 μm filter. The optimal viral titer for each cell type was determined as the least amount of viral supernatant required to transduce 80% of target cells without apparent cytotoxicity.
RT-PCR. Total RNA was extracted from 1×106 transduced cells using Trizol (Invitrogen Life Technologies, Carlsbad, Calif.), and 1 jig of RNA was used for RT-PCR using the Super-Script One-Step RT-PCR system (Invitrogen Life Technologies, Carlsbad, Calif.). Briefly, the cDNAs were synthesized at 50° C. for 30 minutes followed by incubation at 94° C. for 2 minutes. Subsequently, 35 cycles of PCR were carried out with de-naturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds and extension at 72° C. for 1 minute. This was followed by a final incubation at 72° C. for 7 minutes. The sequences of F2-B2 and GAPDH primers were as published (Al-Zoubi et al., (2001), J Biol Chem, 276 (50), 47202-11.). 13L-Forward (5′CGCCGGCGAATCTATGACAAT3′ (SEQ ID NO: 15)) and B2-reverse primers were used to amplify only MADD and IG20pa. The PCR products were then separated on a 2% agarose gel.
Hoechst Staining. 5×105 transduced cells were collected and washed in cold PBS. 1 jig/mL Hoechst 33342 and 5 jig/mL Propidium Iodide (Sigma, St. Louis, Mo.) were used to stain cells for five minutes. Cells with condensed chromatin were analyzed using a BD-LSR (Becton Dickinson, N.J.). Highly PI-positive cells which represent necrotic or late-apoptotic cells were excluded from the analysis. Only GFP-positive cells were included in the analysis.
TMRM staining. 5×105 transduced cells were collected and washed in cold PBS and then stained with 100 nM tetramethylrhodamine methyl ester (Molecular Probes, Invitrogen, CA) for 15 minutes at 37° C. Cells were washed with cold PBS and then subjected to FACS analysis using a FACS Calibur. Only GFP-positive (shRNA-expressing) cells were included in the analysis.
Caspase Detection. 5×105 transduced cells were collected and washed 3× in cold PBS and then stained with either Red-z-VAD-FMK (pan-caspases), Red-IETD-FMK (active caspase-8) or Red-LEHD-FMK (active-caspase-9) (EMD Biosciences Inc.) for 30 minutes at 37° C. Transduced GFP-positive cells were analyzed for active caspase staining using a FACS Calibur.
Cell Proliferation. 24 hours post-transduction, 5×105 HeLa or 8×105 PA-1 cells were plated into six-well plates. Every other day, cells were collected, washed and stained with trypan blue, and trypan blue-negative viable cells were counted.
CFSE Dilution Assay. 24 hours post-transduction, 5×105 HeLa or 8×105 PA-1 cells were stained with 2 μM SNARF-1 carboxylic acid, acetate, succinimidyl ester (S-22801, Molecular Probes, Invitrogen, CA) for 15 minutes at 37° C. Cells were then washed and either used immediately for FACS analysis or plated into six-well plates. Every other day, cells were collected, washed and CFSE dilution, as an indicator of cell division, was determined by FACS analysis.
Crystal Violet Staining. 5×105 HeLa and 8×105 PA-1 cells were plated into six-well plates. 24 hours later cells were treated with different shRNA-expressing lentiviruses for 4 hours. Cells were washed and replenished with fresh warm medium. Twelve days later, cells were fixed in ice-cold methanol and stained with crystal violet to assess viability and colony formation.
Antibodies and other reagents. The anti-IG20 peptide polyclonal antibody, raised against 3 different peptides from the N-terminal, middle and C-terminal region of IG20, has been previously described (Al-Zoubi et al., (2001) J Biol Chem, 276 (50), 47202-11). Anti-Caspase-8 antibody (C-15) was a gift from Marcus E. Peter (Ben May Institute of Cancer Research, University of Chicago, Chicago). Anti-FADD antibodies were obtained from BD PharMingen, San Diego, Calif., and anti-GFP/YFP antibody (JL-8 clone) was purchased from Clontech Palo Alto, Calif. Anti-caspase-8 (C-20) for immunoprecipitation, anti-DR5 (IMG 120), anti-DR4 (H-130), anti-caspase-3 (H-277) and anti-DR4 (B-9 monoclonal) antibodies were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif. Anti-TRAIL-biotinylated antibody and recombinant human-TRAIL were obtained from Peprotech Inc., Rocky Hill, N.J. Anti-actin antibody was obtained from Sigma-Aldrich Corp, CA.
Total RNA was extracted from 1×106 transduced cells using Trizol (Invitrogen Life Technologies, Carlsbad, Calif.), and 1 μg of RNA was used for RT-PCR using the Super-Script One-Step RT-PCR system (Invitrogen Life Technologies, Carlsbad, Calif.). RT-PCR was carried out using F2-B2 and GAPDH primers. The PCR products were then separated on a 2% agarose gel.
FACS analysis of cell surface expression of receptors. Forty-eight hours post-transduction, HeLa cells were collected in enzyme-free cell dissociation buffer (Invitrogen, CA), washed once with PBS containing 0.5% BSA and let stand in the same buffer for 10 minutes at 4° C. PE-conjugated anti-DR4 (DJR1 clone), anti-DR5 (DJR2-4 clone), anti-DcR1 (DJR3 clone), anti-DcR2 (DJR4-1 clone), anti-TRAIL (clone RIK 2) and anti-FasL (NOK1) antibodies purchased from eBiosciences, San Diego, Calif. and anti-Fas (BD Pharmingen) were used for staining samples for 30 minutes at 4° C. A mouse IgG antibody was used as isotype control. Cells were washed with PBS and GFP-positive cells were analyzed by using a FACS Calibur (Becton Dickinson, N.J.).
Suppression of apoptosis using DN-FADD and CrmA. HeLa and PA-1 cells were transfected with either DN-FADD, CrmA or control PCDNA 3.1 vector using Super-Fect reagent (Qiagen Inc., CA). Permanently transfected cells were selected in 800 μg/mL of G418. Post-selection, stably transfected cells were grown in medium containing 400 μg/mL of G418. The stable cells were transduced with the respective lentiviruses and 72 h post-transduction, cells were assayed for spontaneous apoptosis. Cells were treated with 10 ng of TRAIL 36 h post-transduction and assayed for apoptosis by active-caspase-3 staining.
Active-caspase-3 detection by FACS. Active-caspase-3 levels were detected by analyzing PE-positive population using the active-caspase-3-PE staining kit (BD Pharmingen, San Diego, Calif.). Only GFP-positive cells were included in the analysis using a FACS Calibur.
Characterization of DISC immunoprecipitated using anti-DR4/DR5 antibody. HeLa cells (2×107) and PA-1 cells (5×107) transduced for 48 h were collected and washed in cold PBS. Washed cells were lysed in 1 mL of lysis buffer [30 mM Tris/HCl, pH 7.5, 150mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitors cocktail (Roche, Mannheim, Germany), 1% Triton X-100 and 10% glycerol] on ice for 30 minutes and clarified by centrifugation at 12000 rpm for 30 minutes at 4° C. Supernatants were normalized for protein concentration and then immunoprecipitated using 2 μg of H-130 DR4/DR5 antibody on a rotoshaker at 4° C. for 4 h followed by addition of 25 μL of 50% slurry of Protein A/G (Amersham, Piscataway, N.J.) beads for 2 hours. The beads were then washed three times with lysis buffer and boiled in SDS lysis buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50mM DTT and bromophenol blue, pH 6.8) for 5 minutes. Eluates were then subjected to SDS-PAGE using a 12% gel for subsequent immunoblot analysis.
Characterization of DISC immunoprecipitated using anti-TRAIL antibody. HeLa (2×107) cells transduced for 36 h were treated with 250 ng of TRAIL (Peprotech) for 30 minutes either at 4° C. (untreated) or at 37° C. (treated), collected, washed in cold PBS and lysed in 1 mL lysis buffer for 30 minutes on ice. The lysates were clarified and then normalized for protein concentration. The DISC was then immunoprecipitated overnight using 2 μg/mL of anti-TRAIL-biotinylated antibody. The biotinylated antibody was immunoprecipitated with 35 μL of 50% slurry of streptavidin agarose beads. The beads were washed three times with lysis buffer, boiled in SDS sample buffer and separated on a 12% SDS-PAGE gel for immunoblot analysis.
Immunoprecipitation of the MADD complex. HeLa cells were transfected with MADD-YFP. Thirty-six hours post-transduction HeLa (2×107) cells were collected and left untreated or treated with 250 ng/mL TRAIL for 30 minutes at 37° C. At the end of the treatment, cells were washed in cold PBS and pelletted and then lysed in 1 mL of DISC lysis buffer for 30 minutes on ice and clarified by centrifugation. The supernatants were normalized for protein concentration, pre-cleared and were incubated with 10 μL of polyclonal anti-IG20 antibodies on a rotoshaker overnight. The complexes were immunoprecipitated with 50% slurry of protein A/G beads. The beads were washed three times and boiled in SDS lysis buffer for 5 minutes. The eluates were then subjected to SDS-PAGE using a 12% gel for immunoblot analysis.
Immunoblotting. The membranes were blocked in 5% non-fat dry milk in PBS-Tween (PBS with 0.05% Tween 20) for 1 hour. Primary antibodies were used at a concentration of 1 μg/mL and the secondary antibodies were used at a 1:10000 concentration. The blots were developed by enhanced chemiluminescence according to the manufacturer's protocol (Pierce Biotechnology Inc., Rockford, Ill.).
Cell culture and viability assay. HEK293, HeLa and PA-1 cells were cultured. Cell death was determined by Trypan Blue exclusion and the numbers of Trypan Blue-positive and -negative cells were counted using a haemocytometer.
Constructions of MADD Mutants. The serine residues or threonine residues at the consensus Akt phosphorylation sites of MADD were mutated to the alanine residue using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. All constructs were sequenced to ensure that only the desired mutations had been introduced.
Virus Infection and Transfection of Cells. Construction of lentiviruses containing different ShRNAs and virus infection of target cells for MADD knockdown are described elsewhere. The cells were transfected using an Effectene Transfection Kit (Qiagen).
Phosphorylation of MADD In Vitro. Kinase assay was performed using standard procedures. In brief, 2 μg purified GST-tagged wtMADD or MADD mutants were incubated for 1 h at 30° C. with 0.2 μg rAkt, 5 μCi γATP in a kinase buffer. The reaction products were fractionated by SDS-PAGE and 32P-labelled proteins were visualized by autoradiography. Loaded GST-MADD was visualized by probing the same membrane with an anti-MADD antibody.
Metabolic Labeling. HEK293 cells were washed twice with phosphate-free DMEM supplemented with 10% dialyzed FBS and subsequently incubated with 0.3 mCi/ml [32P]orthophosphate in the same medium for 6 h. MADD was immunoprecipitated with an anti-MADD antibody. The precipitated MADD was separated by SDS-PAGE, transferred to a nitrocellulose membrane and subjected to autoradiography. Loaded MADD was visualized by immunoblotting the same membrane using an anti-MADD antibody.
Immunoblot Analysis. Cells were lysed for 1 h at 4° C. in a lysis buffer (20 mM Tris pH 7.5, 2 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol (DTT), 250 mM sucrose, 0.1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100) containing a protease inhibitor cocktail (Sigma, St. Louis, Mo.). Equal protein loading was controlled by Ponceau Red staining of membranes. Blots were probed using corresponding primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, and the protein bands visualized by enhanced chemiluminescence.
Preparation of Subcellular Fractions. Mitochondrial fractions were prepared using standard procedures. Briefly, cells were washed twice with PBS and the pellet was suspended in 0.2 ml of buffer A (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 250 mM sucrose) containing a protease inhibitor cocktail. The cells were homogenized by 12 strokes in a Dounce homogenizer. The homogenates were centrifuged twice at 750 g for 5 min at 4° C. The supernatants were centrifuged at 10000 g for 15 min at 4° C. to collect mitochondria-enriched heavy membranes.
Plasma Membrane Preparations. Plasma membrane and cytosolic fractions were prepared using standard procedures. Cells were washed in ice cold buffer (160 mM NaCl, 38 mM HEPES, pH 7.4, 1 mM MgCl2, 1 mM EGTA) containing protease inhibitor cocktail (Rosch). Cells were lysed by sonication (on setting 5 at 20V) with three pulses of 15 s with 15 s pauses on ice. An aliquot of total cell lysate was removed for western blotting control and the rest was subjected to low speed centrifugation at 400×g, for 10 min at 4° C., to remove unbroken cells, debris, and nuclei. Further the lysate was separated by ultracentrifugation at 117,000×g, 60 min at 4° C. into cytosol and membrane fraction. Membrane pellets were resuspended in 2× Laemlei sample buffer and boiled for 5 min. All samples were frozen at −80° C. Samples were analyzed by western blot and probed with anti-pMADD antibody. Equal loading for membrane and cytosolic fractions were monitored by re-probing the membrane with Caveolin-1 and β-actin, respectively. (Barber M A., et al. JBC, 2007 Oct. 12; 282(41):29967-76).
Immunoprecipitation. Immunoprecipitations were performed using standard procedures. The samples were precleared with 10% (vol/vol) Protein-A agarose (Roche) for 1 h on a rocking platform. Specific antibodies were added and rocked for 1 h. Immunoprecipitates were captured by incubating with 10% (vol/vol) Protein-A agarose for an hour. The agarose beads were spun down and washed three times with NET/NP40 buffer (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 7.5, 0.1% NP-40). The antigens were released and denatured by adding SDS sample buffer. Immunoblots were prepared and analysed as described above.
Generation of Anti-Phospho-S70, -T173 and -T1041 Antibodies. The anti- phospho-antibodies were produced by Eurogentec Deutschland GmbH. Cäcilienstrasse 46. 50667 Köln. GERMANY. The phosphopeptides, CRQRRMpSLRDDTS (SEQ ID NO: 7) (S-70), GSRSRNSpTLTSL (SEQ ID NO: 8) (T-173), and KRKRSPpTESVNTP (SEQ ID NO: 9) (T-1041) were used to immunize the rabbits. The antisera were depleted of antibodies that recognize nonphosphorylated MADD.
Cell Death ELISA and Caspase-8 Activity Assay. Histone-associated DNA fragments were analyzed by a Cell death ELISA Kit (Roche). Caspase-8 activity was detected using the assay kit (R&D System). The assay procedures were followed according to the kit instructions.
Statistical Analysis. Paired data were evaluated by Student's t-test. A 1-way ANOVA was used for multiple comparisons. A value of p<0.05 was considered significant.
An shRNA sequence for “mid” siRNA is GTACCAGCTTCAGTCTTTCTTCAAGAGA GAAAGACTGAAGCTGGTAC (SEQ ID NO: 16), with the hairpin loop region highlighted in bold.
An shRNA sequence for “13L” siRNA is CGGCGAATCTATGACAATCTTCAAGAGA GATTGTCATAGATTCGCCG (SEQ ID NO: 17), with the hairpin loop region highlighted in bold.
Antisense molecules against lab are used in treatments. Sense molecules of lab are used to restore lost lab function in diseased normal cells, for example, gland cells.
The cDNA sequence of MADD splice variant is as follows (SEQ ID NO: 1):
In the MADD cDNA sequence presented above, the highlighted portion in bold indicates nucleotides that are absent in a variation of the MADD splice variant, whose sequence is provided below (SEQ ID NO: 2):
The amino acid sequence of MADD is as follows (SEQ ID NO: 3):